The present invention relates to a novel complex containing a crosslinked avidin, a method for preparing the novel complex, an analyzing method using the crosslinked avidin, an analyzing reagent, and an analyzing kit. The term xe2x80x9canalyzingxe2x80x9d or xe2x80x9canalysisxe2x80x9d as used herein includes a detection used to determine the presence or absence of a compound to be analyzed, and a quantitative determination of an amount of a compound to be analyzed.
An enzyme immunoassay is a highly sensitive assay making use of an antigen-antibody reaction. A typical enzyme immunoassay is a heterogeneous enzyme immunoassay, such as a sandwich method, wherein an antibody immobilized on a solid phase and a labeled antibody are used, or a competitive method wherein the solid phase-immobilized antibody and a labeled antigen are used.
An enzyme-labeled antigen or an enzyme-labeled antibody used in the above assays is prepared by various methods. For example, in an early stage there was developed a method for randomly preparing a polymer of enzymes and antigens or antibodies by adding glutaraldehyde having a bivalent reactivity to a mixture of the enzymes and antigens or antibodies. Thereafter, an improvement was attempted to make use of the fact that peroxidase has a small number of amino groups on the surface thereof, and exhibits a strong resistance to a chemically reactive reagent. More particularly, an improved method for synthesizing only the desired enzyme-labeled antibodies by reacting glutaraldehyde with peroxidase, removing excess aldehyde groups, and mixing the resulting aldehyde-introduced peroxidase with antibodies was developed. Further, a method for producing enzyme-labeled antibodies in a high yield was developed in view of the fact that peroxidase has sugar chains. Specifically, this method comprises oxidizing sugar chains with periodic acid to form aldehyde groups, and binding amino groups of antibodies thereto to produce enzyme-labeled antibodies.
Various crosslinking agents were developed and used in reactions binding antibodies and enzymes. Of these crosslinking agents, a heterobifunctional crosslinking agent having a succinimide group and a maleimide group at both ends was developed. This agent can specifically link an amino group of an enzyme to an SH group in a hinge region of an antibody to produce an enzyme-labeled antibody in a high yield.
Besides the above methods, wherein an antigen or an antibody is bound to an enzyme with a crosslinking agent, coupling methods using an avidin-biotin reaction to bind an antigen or an antibody to an enzyme were developed. In these methods, a combination of two reagents is generally used. More particularly, on one hand, a biotinylating agent is used to prepare an antigen or antibody to which a biotin is introduced, i.e., a biotin-introduced antigen or a biotin-introduced antibody. On the other hand, (1) a complex of an enzyme and an avidin, i.e., an enzyme-labeled avidin, is prepared by a chemically binding method, or (2) a complex of an enzyme and an avidin via a biotin, i.e., an enzyme-labeled avidin, is prepared by mixing a biotin-introduced enzyme and an avidin in a suitable ratio.
The chemically binding method (1) comprises binding an enzyme and an avidin with glutaraldehyde or a heterobifunctional crosslinking agent, and thus can be performed by repeating a procedure of the method for preparing the enzyme-labeled antibody, except that an enzyme and avidin are combined instead of the combination of an enzyme and an antibody.
The biotin-introduced enzyme used in the above method (2) can be prepared, for example, in accordance with a gene manipulation technique, when luciferase is used as an enzyme. Specifically, when a gene containing a luciferase gene and a biotin acceptor gene ligated thereto is expressed in a host cell, a luciferase-biotin acceptor fused protein is synthesized in the host cell. Then, biotin is coupled with the fused protein by an action of the host cell to produce a biotin-introduced enzyme, i.e., a luciferase-biotin acceptor fused protein to which biotin is introduced.
The method wherein the resulting biotin-introduced antigen or biotin-introduced antibody and the enzyme-labeled avidin are used comprises reacting a sample containing a compound (an antigen) to be analyzed with a carrier carrying immobilized antibodies thereon; washing the reaction mixture; reacting the biotin-introduced antibody therewith; washing the reaction mixture; reacting the enzyme-labeled avidin therewith, and measuring an amount of the enzyme stemming from the enzyme-labeled avidin bound to the complex of the immobilized antibodies-carrying carrier and the compound to be analyzed, whereby an amount of the antigen can be determined.
The immunoassay using the avidin-biotin reaction has advantages in that, if the enzyme-labeled avidin is prepared in advance, various antibodies may be used as a label after biotinylation, and in that an enzyme whose activity is heavily damaged by a chemical treatment may be used in the form of an enzyme-labeled avidin while maintaining its activity, if the biotin-introduced enzyme is expressed in accordance with the gene manipulation. However, the above immunoassay has disadvantages in that the number of process steps is increased and data obtained in each determination shows a poorer reproducibility.
An attempt to reduce the reaction steps was made by premixing a biotin-introduced antibody, avidin, and a biotin-introduced enzyme and using them in the form of an enzyme-labeled antibody, i.e., a biotin-introduced antibody-avidin-biotin-introduced enzyme complex. However, the reactivity is greatly lowered, or stability is remarkably reduced, and therefore, it cannot be used as a reagent as it is.
The avidin-biotin reaction is applied, besides the immunoassay, to a DNA or RNA assay by a DNA probe, or a receptor-ligand assay. The application of the avidin-biotin reaction to the above assays has disadvantages similar to those encountered in the immunoassays.
Accordingly, the object of the present invention is to remedy the above-mentioned disadvantages of the prior art, and to provide a quick, convenient and accurate assay having a small number of procedure steps.
The present invention relates to a biotin-avidin-biotin complex comprising at least two biotin-introduced products which are the same or different, and a crosslinked avidin sandwiched therebetween.
In the biotin-avidin-biotin complex of the present invention, at least one of the biotin-introduced products is preferably a biotin-introduced binding component, and at least one of the biotin-introduced products is preferably a biotin-introduced labeling substance.
Further, the present invention relates to a process for preparing the biotin-avidin-biotin complex, comprising the steps of:
(1) treating an avidin with a crosslinking agent to prepare a crosslinked avidin,
(2) biotinylating the same or different substances to be biotinylated to prepare the same or different biotin-introduced products; and
(3) binding the crosslinked avidin and the same or different biotin-introduced products to form the biotin-avidin-biotin complex.
Further, the present invention relates to an analyzing method characterized in that biotin-introduced products which are the same or different, and a crosslinked avidin are used.
Further, the present invention relates to an analyzing method characterized in that (1) a biotin-introduced binding component, (2) a crosslinked avidin, and (3) a biotin-introduced labeling substance are used.
Further, the present invention relates to a method for analyzing a compound to be analyzed characterized in that
(1) a sample possibly containing the compound to be analyzed, a biotin-introduced binding component capable of binding specifically to the compound to be analyzed, a crosslinked avidin, and a biotin-introduced labeling substance are brought into contact with each other in any sequential order, to form a complex of the compound to be analyzed, the biotin-introduced binding component, the crosslinked avidin, and the biotin-introduced labeling substance; and
(2) analyzing a signal derived from the labeling substance in the complex.
Further, the present invention relates to an analyzing reagent characterized by containing a crosslinked avidin.
Further, the present invention relates to an analyzing kit characterized by containing a crosslinked avidin and a biotinylating agent.
Further, the present invention relates to an analyzing kit characterized by containing
(1) a biotin-introduced binding component,
(2) a crosslinked avidin, and
(3) a biotin-introduced labeling substance.
The term xe2x80x9canalyzingxe2x80x9d or xe2x80x9canalysisxe2x80x9d as used herein means a binding analysis wherein the properties of two compounds binding specifically to each other are used, one of the two compounds corresponding to a target compound to be analyzed, and then the target compound is analyzed, using a xe2x80x9cbinding componentxe2x80x9d capable of specifically binding to the target compound to be analyzed. As a combination of the two compounds which specifically bind to each other, there may be mentioned a combination of an antigen and an antibody, a combination of a DNA and a DNA or RNA complementary thereto, a combination of an RNA and a DNA or RNA complementary thereto, a combination of a receptor and a ligand thereof, such as a hormone, cytokine, neurotransmitter, or lectin, a combination of an enzyme and a ligand thereof, such as a substrate analogue of the enzyme, a coenzyme, a regulatory factor, or an inhibitor, a combination of an enzyme analogue and a substrate for an enzyme which is an origin of the enzyme analogue, and a combination of a sugar and a lectin. The enzyme analogue is a compound having a high affinity to a substrate of the original enzyme but exhibiting no catalytic activity.